Abstract Oral pre-exposure prophylaxis (PrEP) with TDF/FTC is an effective prevention intervention for HIV acquisition, in particular when adherence is high [1-4]. However, poor adherence renders this therapy ineffective [5]. Innovations recently introduced into the field of systemic PrEP are long-acting (LA) formulations of antiretrovirals (ARVs) that stably release drugs over many weeks as nano-formulations [6] and have activity in animal models of prevention [7]. To date no systemic LA drug formulations have demonstrated HIV prevention in humans though phase II safety studies of LA Cabotegravir are underway. Injection of these LA ARV are preceded by approximately one month of therapy with the oral formulations to assess for allergy or serious toxicity as once injected these agents have detectable levels for months and the drug cannot be removed or have clearance accelerated. The ultimate goal of this proposal is to address these limitations and develop an injectable polymer-based delivery system for LA PrEP that offers durable and sustained protection from HIV transmission, high efficacy of HIV inhibition, increased user compliance, and the ability to be removed in case of unanticipated adverse events or when considering discontinuation from the LA PrEP. We will achieve this goal by developing a liquid ARV formulation utilizing excipients that form a biodegradable depot after subcutaneous injection (in-situ forming implant (ISFI)). We propose a comprehensive evaluation of this novel drug delivery approach using 1) highly relevant transmitted/founder viruses and highly relevant modes of HIV acquisition via physiologically relevant cell-free and cell-associated viruses, in the presence of human semen using BLT humanized mice; and 2) a highly relevant macaque model of mucosal simian/human immunodeficiency virus (SHIV) as an invaluable preclinical tool to assess the efficacy of the ISFI against SHIV acquisition. This cutting edge combined approach will be utilized to evaluate the scientific premise of our proposal to investigate whether sustained protection against HIV acquisition can be achieved using a unique and highly innovative long-acting coitally-independent antiretroviral ISFI formulation.